domestication of black tiger shrimp (penaeus monodon) in recirculation systems in vietnam

LITERATURE REVIEW ...9 Domestication techniques for black tiger shrimp Penaeus monodon Fabricius, 1798: a literature review.. REARING SYSTEM...35 Effect of water exchange, salinity re

Engineering, Ghent University, Belgium

Local promoter: Dr Nguyen Van Hao

Research Institute for Aquaculture No.2, Ho Chi Minh City, Vietnam

Dean: Prof dr ir Guido Van Huylenbroeck

Rector: Prof dr Paul Van Cauwenberge

Examination Committee and Reading Committee (*):

Prof dr ir Herman Van Langenhove, Chairman, Department of Organic

Chemistry, Faculty of Bioscience Engineering, Ghent University, Belgium

Herman.VanLangenhove@UGent.be

Prof dr Guy Smagghe, Secretary, Department of Crop Protection, Faculty of

Bioscience Engineering, Gent University, Belgium Guy.Smagghe@UGent.be

Prof dr Patrick Sorgeloos, Promoter, Department of Animal Production, Faculty

of Bioscience Engineering, Ghent University, Belgium Patrick.sorgeloos@UGent.be

Dr Nguyen Van Hao, Local VLIR promoter, Research Institute for Aquaculture

No 2, Ho Chi Minh City, Vietnam Haoria2@hcm.vnn.vn

Prof dr ir Peter Bossier, Department of Animal Production, Faculty of Bioscience

Engineering, Ghent University, Belgium Peter.bossier@ugent.be

*Prof dr Geert Janssens, Department of Animal Nutrition, Genetics, Breeding and

Ethology, Faculty of Veterinary Medicine, Ghent University, Belgium

Nguyen Duy Hoa

Domestication of black tiger shrimp (Penaeus monodon) in recirculation systems in Vietnam

Thesis submitted in fulfilment of the requirements for the degree of

Doctor (PhD) in Applied Biological Sciences

Dutch translation of the title:

Domesticatie van de tijgergarnaal (Penaeus monodon) in

recirculatiesystemen in Vietnam

To cite this work:

Hoa, N.D., 2009 Domestication of black tiger shrimp (Penaeus monodon) in recirculation systems in Vietnam PhD thesis, Ghent University, Belgium

The author and the promoters give the authorisation to consult and to copy parts of this work for personal use only Every other use is subject

to the copyright laws Permission to reproduce any material contained in this work should be obtained from the author

ISBN 978-90-5989-342-9

This study was funded by the Flemish Interuniversity Council (VLIR),

ACKNOWLEDGEMENTS

First of all, I am very grateful to the Flemish Interuniversity Council (VLIR) for the scholarship and research grant they provided to complete my PhD-research program Over more than four years, many people were involved in the research of this thesis, all of them I would gratefully like to acknowledge here:

My deepest sincere gratitude is giving to my first promoter Prof dr Patrick Sorgeloos for his enthusiastic guidance, encouragement, and persistent support over four years to this PhD-research work Patrick, behind the success of this thesis was your great scientific and academic contribution Your kind-hearted influence has brought people together to help me finish this research work

I also wish to thank Prof dr ir Peter Bossier for his support to this research work Peter, although you may not have been directly involved in this research work, you strongly welcomed me to do the PhD-research work at the Lab and always followed the progress of my PhD-research work

I am grateful to my local VLIR promoter, Dr Nguyen Van Hao - director of the Research Institute for Aquaculture No.2, for giving me the time to follow the PhD-research work, for his guiding and contributing practical experiences in shrimp farming and for providing facilities, staff and support for the experiments

I am also grateful to Dr Roeland Wouters; he has contributed significantly to the research work on the section of broodstock shrimp nutrition as well as the review of several papers Roeland, without your help I could not have obtained good results on

brother, friend Mathieu, I never forget all what you have done for me You were working hard together with me at the beginning to produce a quality proposal for obtaining a scholarship and research grant You were working hard to help me in sample analysis, in discussion for experimental design, in correction and edition of scientific papers as well as of this thesis

Thanks a lot to Dr Greg Coman from the Commonwealth Scientific and Industrial Research Organization (CSIRO) in Australia who has contributed significantly to the

development of the sand-based recirculation system for our P monodon

domestication program and also for his willingness to share the achieved knowledge from his research program in Australia during my thesis experiments as well as to be

a co-author of one of my papers

Special thanks to Magda Vanhooren, Marc Verschraeghen, Alex Pieters, Caroline

Van Geeteruyen and Dorinda Tack for their constant help in solving administrative

problems Geert Vandewiele and Anita De Haese assisted with the analytical work Jean Dhont, Tom Baelemans, Jorg De Smyter were always willing to help me To Kristof Dierckens, Gilbert Van Stappen, Marijke Van Speybroeck, Brigitte Van Moffaert, Christ Mahieu, Bart Van Delsen, Sebastian Vanopstal, and others, for their friendship and assistance

Thanks a lot many colleagues, Mr Trinh Trung Phi, Mr Vu Thanh, Mr Nguyen Quoc Hung, Mr Tran Kim Dong, Mr Nguyen Hong Bao Duy, Mr Phan Anh Ha, Mr Dao Van Duoc, Mr Ho Phat Cuong, Mrs Le Dung Hoa, Mr Hoang Thanh Lich, Mr

development of this domestication technique

Many thanks to my friends and PhD colleagues, especially Dr Nguyen Thi Ngoc Tinh opened the information of the VLIR scholarship to the young generation at my institute I also wish to thank Dr Le Hong Phuoc, Dr Dang To Van Cam, Dr Nguyen Thi Ngoc Anh, Dr Dinh The Nhan, Ho Manh Tuan, Nhu Van Can, Huynh Thanh Toi, and others, for their support, friendship and making my life colourful during my stay

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

ARA Arachidonic acid

CSIRO Commonwealth Scientific and Industrial Research Organization DHA Docosahexaenoic acid

EFA Essential fatty acid

EPA Eicosapentaenoic acid

FAME Fatty acid methyl esters

FAO Food and agriculture organization

Fo: Wild origin generation

F1: First domesticated generation

F2: Second domesticated generation

HUFA Highly unsaturated fatty acid

ICP-PhD International Course Program-PhD

PCR Polymerase chain reaction

PGs Prostaglandins

PUFA Poly-unsaturated fatty acid

SEAFDEC Southeast Asian Fisheries Development Center

SPF Specific-pathogen-free

VASEP The Vietnam Association of Seafood Exporters and Producers VLIR Vlaamse Interuniversitaire Raad (Flemish Inter-University Council)

Chapter I GENERAL INTRODUCTION 1

Chapter II LITERATURE REVIEW 9

Domestication techniques for black tiger shrimp Penaeus monodon (Fabricius, 1798): a literature review .11

Chapter III REARING SYSTEM 35

Effect of water exchange, salinity regime, stocking density and diets on growth and survival of domesticated black tiger shrimp Penaeus monodon (Fabricius, 1798) reared in sand-based recirculating systems 37

Chapter IV DOMESTICATION PROGRESS 55

Progress in the domestication of black tiger shrimp Penaeus monodon (Fabricius, 1798) in a sand-based recirculation system in Vietnam 57

Chapter V BROODSTOCK NUTRITION 77

Section I A fresh-food maturation diet with an adequate HUFA composition for broodstock nutrition studies in black tiger shrimp Penaeus monodon (Fabricius, 1798) 79

Section II Spawning performance and offspring quality of domesticated black tiger shrimp Penaeus monodon fed a semi-moist maturation pellet 99

Chapter VI GROW-OUT PERFORMANCE 119

Grow-out performance of domesticated Penaeus monodon (Fabricius, 1798) 121

Chapter VII GENERAL DISCUSSION 129

Chapter VIII REFERENCES 147

SUMMARY 169

CURRICULUM VITAE 181

CHAPTER I

GENERAL INTRODUCTION

Chapter I General introduction

The black tiger shrimp, Penaeus monodon Fabricius (1798), is one of the largest

penaeid shrimps in the world, reaching 270 mm in body length (Motoh, 1985), and is

of considerable commercial importance in international markets P monodon is

indigenous to Vietnam and has contributed significantly to the development of the aquaculture sector and hence the economy, with a national production between 350,000-400,000 tons a year, recently According to VASEP (Phi et al., 2009), shrimp farming contributes around 40% in value of the total fisheries export turn-over of the country In 2007, the national shrimp production of approximately 350,000 tons, was

estimated to comprise of 270,000 tons of P monodon and 80,000 tons of white shrimp, Litopenaeus vannamei (Merican, 2008) According to Jeff Jie-Cheng Chuang,

Vice president of Uni-President Vietnam, the annual nationwide demand for postlarvae is around 35 billion (Merican, 2008)

To date, shrimp hatcheries in Vietnam have been able to supply the demands of the commercial shrimp farms, but prevalence of diseases and the low quality of the postlarvae have resulted in huge damage to both the hatchery and grow-out sector Disease outbreaks, especially White Spot Syndrome Virus (WSSV), Yellow Head Virus (YHV), Taura Syndrome Virus (TSV), Monodon BaculoVirus (MBV), etc and

to a lesser extent, luminescent bacterial infections have resulted in a declining global production in recent years for shrimp farmers in Asia and in the America and this leads to an almost inevitable ‘boom-and-bust’ cycle of the shrimp farming industry

(Flegel et al., 2004) Another factor contributing to the unstable development of the shrimp farming industry is the reliance on wild shrimp broodstock for the hatchery industry due to fluctuations in supply and declining natural stocks The fishing of wild broodstock poses serious problems to the natural resource and from an ecological perspective, the fishing of wild stocks moreover is unacceptable in the long term The use of wild broodstock shrimp also poses serious risks for vertical disease transmission from the breeders to the offspring, resulting in disease outbreaks and for this reason, viral diseases must be controlled at the broodstock level itself (Craig, 1998; Yano, 2000; Arce, 2005; Coman et al., 2005)

Successful domestication of Penaeus monodon will ensure sufficient quantities of

good quality broodstock and consequently reliable offspring production The development of reliable domesticated stocks to produce high quality and SPF seed is

therefore imperative to a long-term sustainable development of P monodon

aquaculture With the goal of developing a successful domestication technique to

produce SPF domesticated P monodon stock, this thesis aimed to: i) develop a based recirculation system for the indoor domestication of P monodon, ii) develop a

sand-protocol for each rearing phase of the domestication process in the developed recirculation system; iii) develop suitable broodstock diets and feeding regimes to improve reproductive performance, more specifically fecundity and egg hatching

success, of the domesticated P monodon stock; and iv) evaluate the grow-out

performance of the offspring, produced from the domesticated stocks, at a commercial farm environment The overall goal of this study was to contribute to the sustainable

development of the P monodon farming sector in Vietnam through development of

techniques that could lead to the commercialization of domesticated breeder stocks

The thesis outline can be summarized as follows:

Chapter I (General introduction) draws an outline covering the main points of this

thesis

Chapter II (Literature review) gives an overview of the development of P

monodon domestication techniques during the last decades, focusing on techniques how to close the life cycle of P monodon in captivity, especially, nutritional and

environmental conditions and other zootechniques applied during the domestication process Environmental and seasonal factors, substrate, nutritional requirements, genetic and male effects, as well as endocrinology, which are all known to affect the

quality of domesticated P monodon broodstocks, are being discussed in this review

Knowledge on reproductive biology and maturation of penaeid shrimp are also reviewed in order to better understand the requirements for maturation and

reproduction of penaeid shrimp Knowledge on shrimp nutrition, especially on P monodon and recent studies on broodstock nutrition of P monodon investigating the

specific role of arachidonic acid in maturation, spawning and reproductive performance are also reviewed and discussed in view of quality improvement of

domesticated P monodon stocks

Chapter III (Rearing system) reports on the development of a sand-based

recirculation system through different trials on water exchange regimes, rearing water

salinity, stocking density and diets for the grow-out phase of P monodon

Chapter IV (Domestication progress) developed a protocol for each phase of the

domestication process from eggs to breeders The developed techniques focussed on aspects of the rearing environment and diets for the 3 different rearing phases,

including the grow-out phase (from 1 to 40g), the prematuration phase (from 40 to 80g), and the maturation phase (from 80 to larger than 100g) Performance was evaluated in terms of growth, survival, spawning and reproductive performance

Chapter V (Broodstock nutrition) consists of two parts:

Section I:

A fresh-food maturation diet with an adequate HUFA composition for broodstock nutrition studies in black tiger shrimp Penaeus monodon (Fabricius, 1798) This

section reports on the development of a fresh-food maturation diet for improving the

reproduction of P monodon and which could serve as a basic fresh-food maturation diet for P monodon maturation or as a control diet for further studies on artificial

maturation diets The formulated fresh-food mixture was constructed based on the ratios of ARA/EPA, DHA/EPA and ω3/ω6 fatty acids in mature ovaries of wild P

monodon

Section II:

Spawning performance and offspring quality of domesticated black tiger shrimp Penaeus monodon fed a semi-moist maturation pellet This section describes the development of a feeding regime for optimising maturation and reproduction of P monodon in captivity by testing different substitution levels of the fresh-food

maturation diet (developed in section I) with a recently developed semi-moist maturation diet (BREED-S FRESH®) (INVE Aquaculture N.V., Belgium)

In Chapter VI (Grow-out performance) grow-out performance of domesticated P

monodon postlarvae at different commercial shrimp farms was evaluated and compared with non-domesticated P monodon stocks

Chapter VII (General discussion) reiterates and discusses the overall results of the

experiments and the progress in domestication of P monodon Based on the

discussion, the general conclusions are drawn and prospective research topics are proposed

Chapter VIII (References) contains all the bibliographic citations mentioned in this

thesis

CHAPTER II

LITERATURE REVIEW

Chapter II

Domestication techniques for black tiger shrimp Penaeus monodon

(Fabricius, 1798): a literature review

Nguyen Duy Hoa (1,3) , Mathieu Wille (3) , Roeland Wouters (2) , Nguyen Van Hao (1) , Patrick Sorgeloos (3)

Research Institute for Aquaculture No.2, Ho Chi Minh City, Vietnam(1)

INVE Technologies N.V., Dendermonde, Belgium(2)

Laboratory of Aquaculture & Artemia Reference Center, Ghent university,

Belgium(3)

Abstract

Closing the life cycle of P monodon in indoor rearing systems has recently been

successful with several generations being produced in research programs However,

domesticated P monodon stocks have not yet been commercialized mainly due to

poor reproductive performance, especially low natural mating, low fecundity and poor egg hatching success This paper summarizes present knowledge and current rearing

techniques used for domestication of P monodon for production of

specific-pathogen-free domesticated stocks Environmental and seasonal parameters, tank substrate, nutritional requirements, genetic and male effects, and endocrinological factors are all

known to affect the quality of domesticated P monodon broodstock and are therefore being discussed in more detail A number of selected P monodon domestication

programs, especially those using indoor rearing systems, are reviewed Overall rearing techniques, nutritional and environmental conditions and other zootechniques applied during the domestication process are discussed Knowledge on reproductive biology and maturation of penaeid shrimp is also discussed to elucidate the factors affecting maturation and reproduction of penaeid shrimp Knowledge on shrimp nutrition, with

special focus on P monodon and recent studies on broodstock nutrition of P monodon investigating the specific roles of arachidonic acid in maturation, spawning and reproductive performance of P monodon are also reviewed and discussed

Keywords: domestication, P monodon

1 Introduction

Worldwide expansion of the shrimp farming industry was attributed mostly the success in the production of high quality nauplii through controlled maturation and

reproduction (Browdy, 1992) Several reviews on maturation of penaeid shrimp have been published in the 1980’s and 1990’s (Chamberlain, 1985; Primavera, 1985; Woo, 1988; Bray and Lawrence, 1992; Browdy, 1992; Benzie, 1997; Browdy, 1998) However, all these reviews indicated that the majority of the studies at that time (from the 1970’s to the 1990’s) focused on short-term maturation and breeding technology, rather than full domestication (closing the life cycle from egg to breeder in captivity)

This was especially the case for black tiger shrimp (P monodon) Also in recent

years, a number of reviews on maturation and breeding technology of penaeid shrimp have been published, for example a review on shrimp endocrinology (Huberman, 2000), a review on cultivation of specific pathogen free (SPF) penaeid shrimp broodstock in closed recirculation systems (Yano, 2000) and a review on broodstock shrimp nutrition (Wouters et al., 2001) as well as a review on shrimp larval quality in relation to broodstock condition (Racotta et al., 2003)

Domestication of penaeid shrimp has first succeeded with white shrimp L vannamei

and was subsequently widely developed and commercialized during the 1990’s, spreading from the West into Asia since the 2000’s In contrast, while domestication

of P monodon had also started early in the 1970’s (Aquacop, 1979; Primavera, 1983;

Withyachumnarnkul, 1998), these domestication programs typically started with pond-reared animals (not eggs or larvae), using outdoor ponds without proper

biosecurity and therefore, although some generations of P monodon have been

produced, these programs ultimately proved not successful and were hence not commercialized This might be the reason why no review is found on domestication

technology for P monodon Full domestication of P monodon from egg to breeder in

biosecure closed recirculation tanks in indoor rearing system to produce SPF shrimp only started in the mid 1990’s with several generations of domesticated black tiger

shrimp being produced during the 2000’s (Coman et al, 2005; 2006; 2007b,c)

Domestication of white shrimp (L vannamei) has changed the face of aquaculture since the early 1990’s World shrimp farming production using domesticated L vannamei expanded from only 10% of total shrimp production in 1998 to 75% of total world shrimp production in 2006 (Wyban, 2007) Unlike white shrimp L vannamei, growing up of P monodon in indoor rearing systems to a size suitable to serve as

breeders was not as easy as it might seem; also triggering maturation and optimizing

reproduction of P monodon breeders in captivity proved very difficult Therefore,

while specific-pathogen-free or disease-resistant domesticated stocks of white shrimp

L vannamei have been widely developed and commercialized, the domestication of

P monodon is still largely in an experimental stage due to poor performance of

domesticated broodstocks, especially in terms of egg fecundity and hatching success (Coman et al., 2005; 2006; 2007b)

In the current paper, selected studies on domestication of P monodon are reviewed

and rearing, maturation and breeding techniques discussed in order to draft

recommendations for successful domestication of P monodon in captivity as well as for improvement of the quality of domesticated P monodon stocks The discussion

focuses on aspects of shrimp reproductive biology, tank substrate, environmental conditions, nutrition, endocrinology, male and genetic effects, as well as the

comparison between wild and domesticated P monodon in order to formulate possible solutions for solving the bottlenecks in domestication of P monodon

2 Domestication technique for black tiger shrimp Penaeus monodon

Broadly speaking, domestication can be defined as that condition wherein the breeding, care and feeding of an organism are more or less controlled by man (Hale,

1969) From an agricultural viewpoint, true domestication means: (i) the individual is valued and kept with a specific purpose; (ii) its breeding is subject to human control; (iii) its behaviour is different from that of the wild ancestor; (iv) its morphology (including size and coloration) exhibits variation never seen in the wild; and (v) some

of which would never survive without human protection (Balon, 1995) In aquaculture, domestication is regarded as the acclimatization of an organism to captive conditions with the two key points being rapid growth and a potential for induced spawning in captivity (Hassin et al., 1997) In this paper, domestication is regarded as the full control of the life cycle of the organism from egg to breeder which is capable to produce offspring through generations in captivity

Since the 1970’s several programs have attempted to produce domesticated P monodon breeders whether or not genetically improved and/or specific pathogen free

(SPF) Several research institutes and private companies have been involved, such as Aquacop in Tahiti (French Polynesia), the Southeast Asian Fisheries Development Center (SEAFDEC) in the Philippines, National Center for Genetic Engineering and Biotechnology in Thailand, Department of Fisheries of Malaysia, a commercial program by a French group at Aqualma from Madagascar, a research program by Commonwealth Scientific and Industrial Research Organization (CSIRO) in Australia, and a commercial project by Moana Technologies Inc, which was founded

in Belgium, but operates from Hawaii The following table provides an overview of

published and non-published information of these P monodon domestication

programs:

Table 1 Overview of P monodon domestication programs

Organization/References Domestication characteristics

Aquacop in Tahiti (French

Polynesia) /Aquacop,

1975; 1977; 1979; 1983

Research program by French scientists This program was successfully producing non-SPF captive stocks in earthen ponds and concrete tanks up to the third generation Survival and broodstock performance was good

National Center for

Genetic Engineering and

Biotechnology in Thailand

/ Withyachumnarnkul,

1998

This research program attempted to produce SPF broodstock;

It failed due to the limited biosecurity in earthen ponds Survival and broodstock quality was very low

Aqualma in Madagascar /

Groumellec, 2008

This program is carried out by a French group since 1999 It

is a commercial program to produce SPF domesticated stocks

with high genetic variation, intended to support P monodon

2008, Moana genetically selected SPF stocks have been

commercialized in India, Thailand and Vietnam

Closing the life cycle of P monodon was first succeeded at Tahiti (French Polynesia) during the 1970’s where three generations of domesticated P monodon stocks were

produced (Aquacop, 1977; 1979) This program started with 9 females and 4 males of wild origin, which were kept in 12-m3 tanks with sand substrate A F1 generation was obtained in November 1975 and an F2 generation in August 1976 Postlarvae were reared in 700-m2 earthen ponds at a stocking density of 30-60 shrimp per m2 until they reached 1-2 g size; they were then transferred into 2,500-m2 earthen ponds at a stocking density of 5-30 shrimps per m2 or to concrete tanks of 800 to 1,200m2 at a density of 50 shrimps per m2 where they reached commercial size (15-22 g mean weight) At harvest a few hundreds were stocked in earthen ponds at a density of 2-3 shrimps per m2 until they reached maturation size Daily renewal of the water varied around 10% of the total volume and various artificial pellets and fresh feeds were given to the shrimp The breeders were then transferred into a 400-m2 tank with a sand bottom where flow-through water was injected through perforated plastic pipes which allowed a self-cleaning action and avoided reduction of the substrate Density was 2-4 animals per m2 with a water depth of 2m In this tank, animals were selected

by divers and the largest and healthiest were stocked in 12-m2 circular maturation tanks with 80 cm of water Coral sand was used as tank substrate 200 to 300 % of the total volume was exchanged daily through perforated plastic pipes imbedded in the sand The water temperature fluctuated between 25-29oC, salinity was 34-35g.L-1 and

pH was stable at 8.2 The natural light was reduced 60-90% by shading The maturation diet consisted of fresh troca flesh and a squid-containing pellet with 60% protein given at 15% of the biomass This domestication technology produced domesticated breeders with a fecundity varying between 60,000 and 600,000 eggs, averaging 75,000 eggs for females of 45g and 300,000 eggs for females of 140g

Viability of the eggs was variable; some spawns were totally or partially unfertilized despite the presence of spermmatophores in the thelycum The small size of the males was suspected to be responsible for the variation in fertilization success The broodstock diet made from squid-meal with 60% protein gave the best output

Another intensive research program on P monodon broodstock rearing during the

70’s and 80’s was carried out at the Southeast Asian Fisheries Development Center (SEAFDEC) (Primavera and Gabasa, 1981; Primavera, 1983) This program produced

P monodon broodstock in earthen ponds Over 1 year was needed to produce

broodstocks of 80-100g The maturation of the broodstock was then carried out in a recirculation system, but mortality of the animals in the recirculation system was very high, reaching up to 5.6% per week for males and 9.2% per week for females The broodstock diet given to the shrimp included artificial pellets at 2% (dry weight) and fresh foods (frozen tahong, live annelids, squid, and cow liver) at 5% (wet weight) of the biomass Broodstock shrimp produced by this technology resulted in 100,000 to 600,000 eggs per spawn with an average of 200,000, while hatching success was only between 30.5%-37.5%

In the 1990’s a research program was carried out in Thailand to close the life cycle of

P monodon (Withyachumnarnkul et al., 1998) Pond-reared animals were used as

starting material The program selected the 1% top-sized shrimp from commercial farms weighing around 50-70g These shrimp were screened for specific-pathogen-free (SPF) status The SPF shrimp was stocked in a 1,600-m2 earthen pond at a stocking density of 4-8 animals per m2 for 8 months until the females reached 150-180g and males reached 80-110g Survival of the shrimp after 8 months was only 30% Although the shrimp produced 300,000 eggs per spawn, the hatching success was only around 30%

Another program to develop SPF black tiger domesticated stock under commercial scale started in the late 1990’s in Madagascar (Groumellec, 2008) All the broodstock shrimp were sourced locally in Madagascar and the facilities were designed according

to high biosecurity standards All founding populations passed through a primary and secondary quarantine phase All OIE (Organization of International Epizootics) listed pathogens were tested and the selected population was certified free from these pathogens More than 10,000 wild individuals were collected along the West Coast of Madagascar from 1999 to 2002 The resulting population was established from 198 selected individuals, representing more than 95% of the genetic variability of the base population From there, the breeding program was designed to keep as much as possible the genetic variability within this domesticated population (effective population > 300 at each generation) After the first five generations, the estimated gain in growth was estimated to be 15% per generation, integrating domestication selection, mild directional selection for growth and improvements in rearing methodology

The Malaysian program on SPF black tiger shrimp started in 2001 and this program

produced 3 generations of SPF P monodon broodstock in 2006 (Subramaniam et al.,

2006) The program in Malaysia started with wild collected shrimp, which passed through a primary quarantine and were screened for multiple viral pathogens including White Spot Syndrome Virus (WSSV), Gill-Associated Virus (GAV), Taura Syndrome Virus (TSV), Monodon Baculo Virus (MBV) and Hepatopancreatic Parvo Virus (HPV) The SPF stocks were then transferred to a secondary quarantine area, consisting of maturation and larval/postlarval rearing units to produce F1 progeny Postlarvae of 15 days (PL15) were stocked in outdoor, plastic lined, broodstock grow-out ponds for mass selection and were raised to adult size by maintaining tight bio-

security conditions For family selection, PL28 were stocked in cages till they were 2g These 2g shrimps were tagged and released into grow-out ponds All grow-out broodstock were screened for multiple viruses at two-month intervals until maturity This technology produced animals of 80-100g within 9-12 months These

domesticated P monodon gave on average 200,000 nauplii per spawn Interestingly,

the percentage of good spawns per day of these domesticated animals was improved from an average 1.8% per day in the wild stock to 3.3% per day in F1 and F2 stocks, while weight gain increased 7.3% in F3 as compared to F2 animals and close to 13%

in F2 as compared to F1 animals

A program to fully close the life cycle of P monodon from egg to breeder in

biosecure, indoor rearing systems to produce SPF broodstocks was started by the Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO)

in the late 1990’s (Coman et al., 2005; 2006; 2007b) The program from CSIRO (Coman et al., 2005; 2006; 2007b) similarly started from wild animals, which passed primary and secondary quarantines for selection of SPF broodstock to produce clean progeny for closing the life cycle of the shrimp The SPF 1-g shrimp were stocked in 10m3 sand-based recirculation systems at 10 animals per m2, which was reduced to 4.5 animals per m2 after 5 months and then to 3.5 animals per m2 from the 8th month The program considered the first 8 months of the life cycle as grow-out phase and from 8 months to 11.5 months as maturation phase, after which the animals could then

be used for hatchery work In the grow-out phase the animals were given a diet consisting of 20% squid, 5% bivalve and 70% high protein pellets, while the maturation diet was composed of 30% squid, 20% bivalve, 5% marine worm (polychaete) and the remaining pellets The animals were reared in water of 27.3 ± 0.9oC, salinity of 29 ± 3g.L-1, alkalinity was 80-140mg.L-1, while ammonia NH4+ <

0.7mg.L-1 and nitrite NO2-N < 5mg.L-1 This technology resulted in very good survival (up to 80%) of the shrimp after 11 months in the indoor sand-based recirculation system However, the fecundity and egg hatching success of the animals were much lower as compared to other programs The 11-month old females of 117g produced only 129,780 ± 17,700 eggs per spawn with a hatching success averaging

31.6% Recently, domestication of P monodon in biosecure, controlled environments has led to progress in the selective breeding of P monodon in Australia, where

selectively bred families had higher nauplii production and higher production yield (Preston, 2009)

Since 2000, the Moana project has been able to create a domesticated P monodon

shrimp line and set up a SPF production program through successful selective breeding of the species (Suzy Horemans, manager Moana Vietnam, personal communication) Today, Moana has already a 7th generation grand-parent stock which

is SPF for all important diseases This genetically selected SPF stock has been commercialized in India, Thailand, and Vietnam and the grow-out performance of this stock indicates an improved growth According to Suzy Horemans, trials in Vietnam produced 30g animals in approximately 130 days rearing for Moana postlarvae versus

160 days for wild postlarvae, while this was 120 days versus 140 days in trials in India, respectively The trials in Thailand with Moana postlarvae were similar as in Vietnam, obtaining 30g shrimps after 130 days rearing

In conclusion of the various research programs on domestication of P monodon some

remarkable similarities can be drawn:

1) Strict screening for viruses, passing the shrimp through a primary and secondary quarantine, as well as application of strict hygiene and biosecurity

2) Closing the life cycle of P monodon is possible when starting with SPF stocks

and applying good biosecure conditions Indoor closed sand-based recirculation systems are suitable for maintaining high biosecurity as well as

to provide stable and good environmental conditions (dissolved oxygen,

ammonia, nitrite, etc.) for grow-out and maturation rearing of P monodon 3) The different phases in the life cycle of P monodon should be respected and

the rearing conditions and diet adjusted accordingly in order to provide a suitable rearing environment for each stage

4) High protein pellets and a diversified fresh-food mixture provide a suitable

diet for the grow-out phase of P monodon during domestication in captivity

A stable pH at 8.2 and high salinity (34-35 g.L-1) together with an alkalinity controlled between 80 to 140 mg.L-1 through periodical addition of sodium bicarbonate to the rearing tanks provides good environmental conditions In addition light intensity in the maturation tanks should be reduced up to 90% of the ambient daylight through shading for successful maturation and spawning

of P monodon

5) Although high protein pellets and a diversified fresh-food mixture were

adequate for grow-out of P monodon from egg to breeder of 100g, low

performance of the domesticated broodstock in terms of natural mating, fecundity and hatching success still remains a bottleneck for the

commercialization of domesticated P monodon stocks More broodstock

nutrition studies are therefore needed to improve the quality of the domesticated broodstock

6) Domestication in conjunction with selective breeding resulted in considerable

improvement of growth of domesticated P monodon through succesive

generations (7.3-13% in the Malaysian program and 15% in the program in Madagascar)

It remains unclear why reproductive performance of domesticated P monodon stocks

was lower as compared to their wild counterparts, especially in terms of natural

mating success, fecundity and hatching success, and why domestication of P monodon was more difficult than for L vannamei In order to help explain this and

formulate ways to improve domestication techniques in the future, in the following sections, several factors that affect maturation and reproduction of penaeid shrimp in

general, and P monodon in particular, are discussed in more detail:

2.1 Environmental conditions

Several studies cited by Chamberlain (1985) indicated the importance of environmental and seasonal factors in shrimp maturation and reproduction In the marine environment, penaeid shrimp often migrates to species-specific depth zones (Perez-Farfante, 1969) Therefore, parameters such as water hydrostatic pressure, light intensity and spectrum related to depth could affect the reproductive biology of penaeid shrimp Hydrostatic pressure has been documented to affect the behaviour of planktonic decapod larvae, but rarely affecting benthic adults (Hardy & Bainbridge, 1951; Knight-Jones & Morgan, 1966) It was also noted that some penaeid shrimp can spawn naturally without eyestalk ablation in shallow water conditions or in race-way systems Therefore it seems unlikely that hydrostatic pressure plays an important role

in reproduction Light intensity and light spectrum are considered two factors related

to depth that affects penaeid reproduction These two factors are related to cloud cover and diurnal and seasonal variation in illumination and to variations caused by dissolved and suspended matter and may account for fluctuations in spawning of wild

decapods to both light intensity and spectrum (Herman, 1962; Segal, 1970; Goldsmith, 1972) Studies by Chamberlain and Lawrence (1981) and Wurts and

Stickney (1984) on Litopenaeus vannamei and Penaeus stylirostris shrimp indicated

positive effects of blue or green light with intensity less than 12 µWcm-2 on

maturation and spawning Rearing water used for domestication of P monodon is

usually clear because of sand filtration High light intensity inhibits maturation in penaeid shrimp and is stressful to the broodstock, therefore a dark environment

created by the reduction of light intensity is essential for P monodon, L vannamei and P japonicus broodstocks and even within the same species the optimum light

intensity for maturation tank might slightly differ for each population caused by differences in depth and turbidity conditions (Yano, 2000) According to Primavera (1983, 1985) reduced light intensity by covering the tanks speeded up the maturation process and prevented stress on black tiger breeders Emmerson (1980) indicated the black coloured tanks resulted in two times more spawns as compared to bright colour tanks

Seasonal peaks in reproduction are characteristic of penaeids, even in tropical species which may exhibit some degree of reproductive activity throughout the year (Lindner

& Anderson, 1956; Subrahmanyam, 1965; Rao, 1968; Pillay & Nair, 1971; Badawi, 1975) Annual spawning peaks, which are synchronized within a species and often variable among species, are probably entrained primarily to photoperiod and/or temperature (Giese, 1959) However, in tropical areas where annual variations in photoperiod and temperature are minimal, salinity may serve as the seasonal reproductive cue; particularly if pronounced wet and dry seasons exist (Pillay & Nair, 1971) Aragón (2007) studied the relationship between water temperature and

spawning season of Penaeus stylirostris in a lagoon in California and found a

spawning peak from May to August when the temperature of the surface-water was

higher and less variable Adult P monodon migrate to the deep sea for maturation and

spawning where environmental parameters such as salinity, pH, temperature, ammonia and nitrite are known to be very stable and/or low, which reduces stress as well as provide a quite environment for inducing maturation and spawning Similar environmental conditions should be provided to captive breeders According to

Ruangpanit et al (1984), P monodon caught from their spawning grounds in the

Indian Ocean where salinity was 33g.L-1 resulted in better maturation and egg fertilisation as compared to shrimp caught from Songkhla lake where the salinity was 22-28g.L-1 High salinity and pH similar to open ocean must be maintained for maturation; however, lower salinity (15-25 g.L-1) seems best to stimulate growth in the grow-out phase (Yano, 2000; Queensland Department of Primary Industries and Fisheries, 2006) Maintaining pH stable at around 8.2 by periodically adding sodium bicarbonate into the tanks is considered good for maturation, spawning and hatching success (Primavera, 1985) An environment with stable water temperature, low concentrations of ammonia and nitrite, etc therefore should be created for successful

domestication of P monodon Closed systems with application of recirculation

technology are considered to meet these requirements

2.2 Substrate and stress

Adult penaeid shrimp migrate from coastal areas to deeper sea for maturation and for natural mating and spawning Specific depths and substrate types are preferred as

spawning grounds, which largely differ from species to species The shrimp Penaeus duorarum prefers depths of 26-60m and a sandy mud bottom containing more than

50% fine sediment (<50µm) with a high organic content (Garcia and L’home, 1980),

suitable for this species (Motoh, 1985) Most successful domestication programs for

P monodon used sand as substrate for the rearing tanks Survival of the shrimp in

those programs using sand substrate was very high (Aquacop, 1979; Coman et al., 2005) Similar results were obtained when artificial substrate such as Aquamat® was added (Arnold et al., 2006) Shrimp broodstock need a quiet environment, without excess food, noise, or human disturbance (Yano, 2000) Therefore, maturation tank rooms should be isolated completely from other rooms Maturation of shrimp is also strongly affected by stocking density because high stocking density causes poor water quality, e.g high ammonia levels, and high levels of stress due to the behavioural interaction of the shrimp (Yano, 1993)

2.3 Nutrition

In contrast to white shrimp L vannamei, only recently progress has been made to unravel the nutritional requirements of P monodon A high protein level of 40-50%, a

lipid level of 4-11%, 1% cholesterol and 0.5-1% n-3 high polyunsaturated fatty acids

in the diet are thought to maintain optimum growth (Shiau, 2008) Higher lipid levels (7-14%) might be required in broodstock diets (Meunpol, 2005b) as ovarian lipid content progressively increased through maturation stages and lipids of maturing females accumulate in the hepantoceas is known to be transferred to the ripening

ovaries and finally to the eggs (Millamena et al., 1993) For broodstock diets of P monodon a protein level of at least 50% may be required (Marsden et al., 1997) In some domestication programs the broodstock pellet for P monodon even contained

up to 60% protein (Aquacop, 1979) High dietary protein levels normally support

growth rather than maturation The high protein requirement of P monodon breeders

is therefore species specific or it may indicate a specific requirement for specific

maturation and reproduction The high protein requirement of P monodon was not taken into account in several of the early domestication attempts of P monodon This

could be a reason for the high mortality and low growth of the species in these

studies In commercial grow-out ponds, P monodon can grow and develop well on

grow-out pellets containing only 36-40% protein; however in these, besides the pellets provided by the farmer, the animals feed on other feed sources such as algae, bacterial substrates or other organisms available in the pond In contrast these extra protein sources are not or only in very limited amounts available in indoor rearing systems, especially in clear water systems Therefore, high protein diets should be provided to

P monodon in indoor rearing system From the early studies, only the domestication

program of Aquacop used a pellet feed with high protein level (60%) Results of this program were the best in the 1970’s The more recent domestication program at CSIRO also fed the shrimp a high protein pellet (55%) This resulted in a very good growth and survival after 11 months (up to 80% survival and the animals reached 120g within 11 months) However, these research programs still encountered poor performance of the domesticated broodstocks in terms of fecundity and egg hatching success The large fraction of grow-out pellets in the diet (70% until month 8 and 45% during the maturation phase from month 8 to month 11) together with the incompletely shading of the maturation tanks were however suspected to be responsible for the low fecundity and egg hatching results in the CSIRO program Improper nutrition is known to prevent shrimp gonad maturation (Aquacop, 1977) and perhaps even affect larval viability According to several studies cited by Chamberlain (1985), the chief component of crustacean egg yolk is a high-density, lipoglycoprotein, termed crustacean lipovitellin, which contains 27-35% lipid, a small carbohydrate component, and no protein-bound phosphorus (Zagalsky, 1976) The